1. Field of the Invention
This invention relates to a measuring apparatus for measuring enzyme reaction rate on the basis of the detected information of the intensity of fluorescence such as increasing rate of the intensity of fluorescence emitted from the fluorescent materials produced by enzymatic reactions, and more particularly to a measuring apparatus for enzyme immunoassay for favorably determining the quantity of the biological active materials.
2. Description of the Related Art
Conventionally, as one of enzyme assays to detect or measure the quantity of enzyme, one method is known to measure the rate of enzyme reactions on the basis of detecting decreasing rate of photo absorption and fluorescence of enzyme substrates in the sample derived from enzyme reactions or increasing rate of photo absorption and fluorescence derived from enzyme reaction products. Generally, a method to detect the absorption of light is used in most cases. However, fluorometry is often used for measuring a very small quantity of enzyme.
Especially, in an automated equipment for immunoassay which has been introduced recently with the aim of detecting or determining micro constituents in biological samples, that is, in the equipment on the basis of so called enzyme immunoassay, wherein very small quantity of physiologically active materials is linked with enzyme by immunoreactions, and the linked quantity of enzyme is measured, fluorometry is used in most cases for the necessity of measuring very small quantity of enzyme.
In the fluorometry, for example, in order to measure the quantity of fluorescent materials, the fluorometer is generally used, wherein included are an applied light system to excite samples including the fluorescent materials for measuring such as the quantity of the fluorescent materials, a measuring system of the received light to detect and measure the intensity of fluorescence by receiving the fluorescent light emitted from the fluorescent materials, and a data processor to determine the rate of the enzyme reaction from the variations of the intensity of fluorescence as the actual information.
In the aforementioned conventional apparatus for measuring the rate of enzyme reactions, especially the data processor thereof, it is common to measure the quantity or density of the fluorescent materials by using a known conversion equation on the basis of data (the intensity of fluorescence) obtained from actual measurement through the aforementioned measuring system of the received light, and further from the quantitative variations (or the variations in density), to finally calculate the reaction rate of enzyme through linear regression using a method of least squares. However, in this kind of measurement, some problems may occur due to the error in measured rate of enzyme reactions caused by the absorption of the excited light into fluorescent materials.
When the density is low in fluorescent materials and under the conditions where there are fairly few degrees of the absorption of the excited light by the fluorescent materials, it may be said that there will be few problems on the errors in the measured results of the rate of enzyme reaction calculated by the aforementioned formula, but on the other hand, under the conditions where the density of fluorescent materials is high and the ratio of the absorption of the excited light becomes larger than several percentages, it will be difficult to determine the exact measurement values by calculating the rate of enzyme reactions from the intensity of fluorescence on the basis of the aforementioned linear equation, thereby necessitates the elimination of the errors caused by the absorption of the excited light for obtaining the exact results of the measurement.